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[alpha]-Hederin inhibits G protein-coupled receptor kinase 2-mediated phosphorylation of [[beta].sub.2]-adrenergic receptors.


Background: Recently is has been shown that [alpha]- and [beta]-hederin increase the [[beta].sub.2]-adrenergic responsiveness of alveolar type II cells (A549) and human airway smooth muscle cells (HASM), respectively, by inhibiting the internalization of [[beta].sub.2]-adrenergic receptors ([[beta].sub.2]AR) under stimulating conditions. Internalization of [[beta].sub.2]AR is initiated by phosphorylations of certain serines and threonines by cAMP dependent protein kinase A (PKA) and G protein-coupled receptor kinases (GRK).

Purpose: To evaluate the effect of [alpha]-hederin on PKA and GRK2 mediated phosphorylation of GFP-tagged [[beta].sub.2]AR.

Study design: To study this process we performed In-Cell Western using isoprenaline stimulated HEK293 cells overexpressing [[beta].sub.2]AR as GFP fusion protein and specific antibodies against PKA (Ser345/346) and GRK2 (Ser355/356) phosphorylation sites.

Results: There was no effect found on the PKA mediated phosphorylation (n = 14) but we could show that [alpha]-hederin (1 [micro]M, 12 h) significantly inhibits GRK2 mediated phosphorylation at Ser355/356 by 11 [+ or -] 5% (n [greater than or equal to] 29, p [less than or equal to] 0.01) under stimulating conditions compared to the positive control. In Forster resonance energy transfer (FRET) experiments using the isolated kinases in solution [alpha]-hederin did not show any influence neither to GRK2 nor to PKA.

Conclusion: Taken together, these results indicate that [alpha]-hederin acts as an indirect GRK2 inhibitor leading to a reduced homologous desensitization of [[beta].sub.2]AR-GFP in HEK293 cells


[[beta].sub.2]-adrenergic receptor



homologous desensitization



Dry extracts from Hedera helix Linne, Araliaceae leaves are well established in the treatment of acute and chronic diseases of the respiratory tract associated with coughing. Their effects are described as bronchospasmolytic and secretolytic and are proven by spirometric and body plethysmographic investigations in randomized placebo-controlled clinical trials and post-marketing surveillance studies (Gulyas et al, 1997; Hofmann et al? 2003; Kraft, 2004; Mansfeld et al., 1997, Mansfeld et al? 1998).

Saponins are considered to be pharmacologically active compounds of H. helix leaves dry extracts by increasing the [[beta].sub.2]-adrenergic responsiveness of alveolar type II cells and bronchial muscle cells, respectively. Among them, [alpha]- and [beta]-hederin which make up approx. 1% and 0.1%, respectively, depending on the extraction procedure inhibit the internalization of [[beta].sub.2]-adrenergic receptors ([[beta].sub.2]AR) under stimulating conditions followed by an increased [[beta].sub.2]AR binding and an elevated cAMP production in vitro (Greunke et al., 2015; Sieben et al., 2009).

An elevated intracellular cAMP level causes the intensified production and secretion of surfactant from alveolar type II cells (A549), liquefying viscous mucus associated with diseases of the respiratory tract. In bronchial muscle cells the increased cAMP level leads to protein kinase A (PKA) mediated phosphorylation of the myosin light-chain kinase (MLCK) which prevents the formation of the MLCK-calmodulin/[Ca.sup.2+] complex required for muscle contraction (Giembycz and Newton, 2006). Additionally, the intracellular [Ca.sup.2+]-level decreases due to influx inhibition through calcium channels and inhibition of release from intracellular stores (Johnson, 1998). These effects provide the biochemical basis to explain the secretolytic and bronchospasmolytic properties of H. helix leaves dry extracts seen within patients in clinical studies (Mansfeld et al., 1997; Mansfeld et al., 1998).

To date it is not clear in which manner [alpha]- and [beta]-hederin inhibit the internalization of [[beta].sub.2]AR. However, it is established that the internalization process is initiated by phosphorylation of the [[beta].sub.2]AR. This desensitization is either mediated by phosphorylation of Ser345/346 and Ser261/262 by PKA (heterologous desensitization) or by phosphorylation of certain serines, especially Ser355/356 in the C-terminus of [[beta].sub.2]AR by G-protein coupled receptor kinase 2 (GRK2) (homologous desensitization) (Tran et al., 2004). The desensitized [[beta].sub.2]AR is subsequently redistributed to a coated pit and finally internalized in form of early endosomes by endocytosis (Ferguson, 2001).

H. helix leaves contain several monodesmosidic saponins in which [alpha]-hederin dominates quantitatively (Fig. 1).

In the present paper our approach was to investigate the influence of [alpha]-hederin on the desensitization of [[beta].sub.2]AR overexpressed as green fluorescent protein (GFP) fusion protein in human embryonic kidney 293 (HEK293) cells by means of In-Cell Western. Bisdesmosidic saponins like hederacoside C and corresponding aglycones like hederagenin were not further investigated because they did not influence the internalization of [[beta].sub.2]AR (Sieben et al., 2009).

Materials and methods


[alpha]-Hederin was obtained from HWI-Analytik (Rulzheim, Germany, batch no. 301174836, m/z = 750,98 for [C.sub.41] [H.sub.66] [O.sub.12]). Purity of [alpha]-hederin was investigated by HPLC (Waters, Eschborn, Germany) equipped with a HPLC pump (MSDS 600 E), an autosampler (WISP 700), a diode array detector (996) and a four-channelonline degasser (Knauer, Berlin). The MILLENNIUM V2.1 software was used for data analysis and processing. The following parameters were used: sample preparation: 5.20 mg [alpha]-hederin dissolved in 25.00 ml methanol, column: LiChrospher[R] 100 RP18, 5 [micro]m, 125 x 4 mm (Merck, Darmstadt, Germany), diode array detector: 205 nm, injection volume: 20 [micro]l, mobile phase A: dest. water/acetonitrile = 44/2 (v/v); adjust to pH 2 using phosphoric acid (85%), mobile phase B: acetonitrile, gradient: 0-9 min 100% A, 9-10 min to 6% B, 10-25 min to 15% B, 25-50 min to 60% B, 50-51 min to 100% B. Retention time of [alpha]-hederin was 48.8 min. Purity of [alpha]-hederin was 98.6%. All other reagents were obtained from Merck (Darmstadt, Germany), if not stated otherwise.

Cell culture

HEK293 cells obtained from DSMZ (No. ACC 305) (Braunschweig, Germany) were cultivated in DMEM medium (Life Technologies, Carlsbad, CA) supplemented with 100 units/ml penicillin, 100 [micro]g/ml streptomycin, and 10% fetal calf serum. Cells were used for experiments after reaching a confluence of 70-80%.

Transfection of HEK293 cells

The [[beta].sub.2]AR-GFP construct was a kind donation of M. J. Lohse (Department of Pharmacology and Toxicology, University of Wurzburg, Germany). The cloning procedure of the [[beta].sub.2]AR-GFP plasmid and the transfection of the HEK 293 cells were described previously (Kallal et al., 1998; Sieben et al., 2009).

Live cell imaging

Pictures of live HEK293 cells stably expressing [[beta].sub.2]AR-GFP were taken with an Axiovert[R] 200 M (Zeiss, Jena) fluorescence microscope equipped with argon laser, beam splitter HFT 488/543, oil immersion objective (Plan Apochromat 63x/1.4), bandpass filter BP 505-530, and pinhole 96 mm was used. Pictures were recorded with an AxioCam[R] (Zeiss, Jena, resolution of 512 x 512 pixels) camera. The camera was controlled by the Axiovision[R] (Zeiss, Jena, release 4.7) software. For internalization experiments, cells were incubated for 30 min. with 10 [micro]M terbutaline hemisulfate in Locke's solution (154.0 mM NaCl, 5.6 mM KC1, 2.3 mM Ca[Cl.sub.2 ]dihydrate, 1.0 mM Mg[Cl.sub.2 ]hexahydrate, 3.6 mM NaHC[O.sub.3], 5.0 mM HEPES, and 2.0 mM D-(+)-glucose monohydrate, pH 7.3). All pictures were taken at 20[degrees]C.

Western blot

For Western blots HEK293 cells stably expressing [[beta].sub.2]AR-GFP were starved overnight in serum-free cell culture medium. They were washed with ice cold PBS (137 mM NaCl, 10 mM [Na.sub.2]HP[O.sub.4] x 2[H.sub.2]O, 2.7 mM KC1, 2 mM [K.sub.2]HP[O.sub.4], pH 7.4) and resuspended with solubilization buffer (150 mM NaCl, 20 mM HEPES, 20 mM [Na.sub.4][P.sub.2][O.sub.7], 10 mM NaF, 10 [micro]g/ml leupeptin, 10 [micro]g/ml phosphatase inhibitor cocktail 2, 10 [micro]g/ml protease inhibitor cocktail 2, 10 [micro]g/ml trypsin inhibitor, 0.9% n-dodecyl-[beta]-D-maltoside) for 30 min. in motion at 4[degrees]C. Cell debris was centrifuged at 20,000 g for 15 min. Partly, the lysate was used for the protein determination according to Bradford using the BioRad protein assay Kit II (BioRad Laboratories, Herkules, CA). The remaining lysate was diluted with native sample buffer (50 mM Tris-HCl pH 6.8, 40% glycerol, 0.01% bromophenol blue), and applied equally to a Native PAGE gel containing 4% polyacrylamide in the stacking gel and 6% polyacrylamide in the resolving gel, respectively. The proteins were stacked at 40 V and separated at 90 V in the resolving gel and subsequently incubated for five min. in 1% SDS solution before it was blotted in the semi-dry method to PVDF membrane with 3.5 mA/[cm.sup.2] for 2 h. The membrane was washed with water and blocked overnight at 4[degrees]C with Odyssey blocking Buffer (LI-COR Biosciences, Bad Homburg, Germany). Finally, the membrane was incubated with the primary antibodies in Odyssey blocking buffer containing 0.1% Tween 20 for 1.5 h. To confirm the stable transfection of HEK293 cells a rabbit antibody against the C-terminus of the [[beta].sub.2]-adrenergic receptor was used (Santa Cruz Biotechnology Inc., Santa Cruz, CA, sc-9042, diluted 1:1,000). Additionally antibodies against the phosphorylation sites Ser 345/346 for PKA (Santa Cruz Biotechnology Inc., sc-16718-R, diluted 1:500) and Ser 355/356 for GRK2 (Santa Cruz Biotechnology Inc., sc-16719-R, diluted 1:500) were used to prove the quality of the antibodies used in the In-Cell Western. After five washing steps with 0.1% Tween20 in PBS, the membranes were incubated with a secondary goat anti rabbit IRDye800 coupled antibody (Ll-COR Biosciences, Bad Homburg, Germany, 926-32211, diluted 1:50,000) for 1 h. After five washing steps with PBS detection was done in the Odyssey plate reader at 700 nm and at 800 nm.

In-Cell Western

The In-Cell Western was performed in 96-well plates which were coated overnight with PDL solution and seeded with 15,000 cells per well. The HEK293-[[beta].sub.2]AR-GFP cells were grown to a confluence of about 80% and starved overnight in serum-free culture medium. Simultaneously pre-incubation with [alpha]-hederin was performed overnight. For negative controls cells were either incubated for 20 min. with 1 [micro]M GRK2-Inhibitor methyl-5-[2-(5-nitro-2-furyl)vinyl]-2-furoate (lino et al., 2002) (EMD Chemicals, NJ) or for 1 h with 7.5 [micro]M PKA inhibitor H89 (Lochner and Moolman, 2006). Stimulation was done with 0.25 [micro]M or 0.5 [micro]M isoprenaline for 20 min., except for the unstimulated control cells. To prevent dephosphorylation cells were fixed with 4% PFA solution and 15% sucrose. All following steps were performed at room temperature and under slight movement. After permeating five times with 0.1% Triton-X 100 in TBS the remaining unphosphorylated receptors were blocked with Odyssey[R] blocking buffer (LI-COR Biosciences, Bad Homburg, Germany, P/N 927- 40000) for 1 h hour. Incubation with the primary antibodies against the PKA (sc-16718R rabbit 1:200) and GRK2 phosphorylated receptor (sc-16719-R rabbit 1:200) was performed for 2.5 h. After five washing steps with 0.1% Tween-20 in PBS the secondary antibody, coupled to IRDye800 (LI-COR Biosciences, Bad Homburg, Germany, IRDye-GAR goat anti rabbit, P/N 926-32211, diluted 1:800), was added for 1 h in the dark. An additive of Draq5 (DNA stain) and Sapphire700 (unspecific cell stain) was used for cell number normalization. Nuclear staining control was performed without Draq5 (Biostatus, Leicestershire, UK) and Sapphire700 (LI-COR Biosiences). After further washing steps on the Odyssey[R] Imager (LI-COR Biosciences) detection took place using Odyssey[R] Application Software 3.0.21 at 700 and 800 nm. Data were analyzed with an In-Cell Western Plug-In from LI-COR. The results are shown as the integrated intension at 800 nm, which correlates with the phosphorylation intensity, normalized to the cell count measured at 700 nm and corrected for the background, measured in cells without staining.


Forster resonance energy transfer (FRET) experiments were performed in 384-well microplates (Greiner Bio One International GmbH, Kremsmunster, Austria) using the Z'-Lyte[TM] kinase assay Kit Ser/Thr 1 (Life Technologies) for the PKA mediated phosphorylation and the Z'-Lyte[TM] kinase assay Kit Ser/Thr 16 (Life Technologies) for the GRK2 mediated phosphorylation, respectively. ATP/substrate mixture (1 nM substrate, 400 nM ATP, 6 nM cAMP) dissolved in kinase buffer (250 mM HEPES (pH 7.5), 50 mM Mg[Cl.sub.2], 5 mM EGTA, 0.05% Brij-35) was placed to each well first and subsequently phosphorylated after addition of the corresponding kinase for 1 h. Kinases dissolved in 0.5% DMSO were used in concentrations of 0.63 [micro]M (50 ng/[micro]l) for GRK2 and 1.15 [micro]M (50 ng/[micro]l) for PKA. Development reagent diluted with development buffer was added and the plate was further incubated for 1 h. The reaction was terminated by adding stop solution (Proprietary reagent, Cat. Nr. P3094). The reaction mixtures were measured on a Tecar[R] Genius Reader (Tecan, Maennedorf, Switzerland) at 460 nm and 535 nm. The results are shown as emission ratio of fluorescence intension at 460 nm to 535 nm normalized to the solvent control (0.5% DMSO). As positive control for both assays, the broad spectrum kinase inhibitor staurosporine dissolved in 0.5% DMSO was used in a concentration of 50 [micro]M for GRK2 inhibition and 20 [micro]M for PKA inhibition (Ruegg and Burgess, 1989). Emission ratio of fluorescence intension at 460 to 535 nm for unphosphorylated substrate was determined in an approach without kinases. [alpha]-Hederin was tested in a concentration of 1 [micro]M.

Statistical data evaluation

Statistical data evaluation was performed with one factorial analysis of variance (ANOVA) after their normal distribution was ensured by means of a D'Agostino & Pearson normality test. Dunnett's post hoc test was subsequently performed. Results were considered to be significant for p values of < 0.05.


Terbutaline-induced internalization and detection of isoprenaline-induced phosphorylation of [[beta].sub.2]AR-GFP

HEK293 cells overexpressing the [[beta].sub.2]AR-GFP were used to study the phosphorylation of [[beta].sub.2]AR under stimulating conditions.

GFP-tagged [[beta].sub.2]AR function was proven by stimulation with 10 [micro]M isoprenaline for 30 min. and a subsequent pronounced endocytotic [[beta].sub.2]AR-GFP internalization, which was recognized by large intracellular vesicles (Fig. 2A and Video 1 Supplemental material).

The presence of [[beta].sub.2]AR-GFP and both GRK2 mediated [[beta].sub.2]AR phosphorylation at Ser 355/356 and PKA-mediated [[beta].sub.2]AR phosphorylation at Ser 345/346 in isoprenaline stimulated HEK293-[[beta].sub.2]AR-GFP cells were demonstrated by Western blots (Fig. 2B). As expected, the receptor and its two phosphorylated forms showed a similar migration in the running gel which also demonstrated the functionality of the primary antibodies (Fig. 2B).

Effect of [alpha]-hederin on the PKA and CRK2 mediated phosphorylation of [[beta].sub.2]AR-GFP in HEK293 cells

The influence of [alpha]-hederin on the phosphorylation of [[beta].sub.2]AR by GRK2 and PKA was investigated in concentrations between 0.1 [micro]M and 1 [[beta].sub.2]M using the In-Cell Western method. Stably transfected HEK293-[[beta].sub.2]AR-GFP cells were incubated for 20 min. with 0.25 [micro]M isoprenaline as positive control which led to a statistically significant increase of PKA mediated phosphorylation at Ser 345/346 of 20.6 [+ or -] 6.4% (n [greater than or equal to] 12, p [less than or equal to] 0.001) compared to untreated control cells. Pre-treatment with 7.5 [micro]M H89 (PKA inhibitor) for 60 min. showed a statistically significant decrease of the isoprenaiine induced phosphorylation of 18.2 [+ or -] 7.6% (n [greater than or equal to] 14, p < 0.05) compared to the positive control, whereas pre-treatment with 1 [micro]M [alpha]-hederin for 12 h showed no influence (n = 14) (Fig. 3A).

Cells stimulated with 0.5 [micro]M isoprenaiine for 20 min. (positive control) revealed a statistically significant increase of GRK2mediated Ser 355/356 phosphorylation of [[beta].sub.2]AR of 30.0 [+ or -] 4.1% (n [greater than or equal to] 29, p < 0.001) compared to untreated control cells. Pre-incubation with 1 [micro]M of methyl-5-[2-(5-nitro-2-furyl)vinyl]-2-furoate (GRK2 inhibitor) statistically significant decreased the isoprenaiine induced phosphorylation by 19.7 [+ or -] 5.3% (n = 29, p < 0.001) compared to the positive control (Fig. 3B). Pre-treatment with [alpha]-hederin in a concentration range between 0.1 [micro]M and 1 [micro]M led to a dose-dependent inhibition of the isoprenaiine induced and GRK2-mediated [[beta].sub.2]AR-GFP phosphorylation. Under stimulating conditions 1 [micro]M [alpha]-hederin inhibited the Ser 355/356 phosphorylation statistically significant with 11.1 [+ or -] 5% (n [greater than or equal to] 29, p < 0.01) compared to the positive control (Fig. 3B).

Effect of [alpha]-hederin on the PKA and CRK2 activity in solution

The direct influence of [alpha]-hederin on the GRK2 activity in solution was studied with the Z'-Lyte[TM] kinase assay Kit 16. The measurement is based on a FRET signal of a coumarin and fluorescein double-labeled protein (substrate) under proteolytic conditions. GRK2 dissolved in 0.5% DMSO (solvent control) in a concentration of 0.63 [micro]M prevents the proteolytic cleavage by phosphorylation of the substrate and the corresponding FRET signal calculated as emission ratio at 440 nm and 535 nm ([Em.sub.440/535]) normalized to the value 1.0 [+ or -] 0.08 (n = 19). Compared to the pure GRK2 approach DMSO showed no change in the GRK2 activity (n = 18) (Fig. 4A). The applied substrate revealed a relative increase of the [Em.sub.440/535] value by a factor of 1.71 [+ or -] 0.23 (n = 19) after maximum proteolytic cleavage. Fifty [micro]M of the broad spectrum kinase inhibitor staurosporine served as positive control. Compared to GRK2 dissolved in 0.5% DMSO the [Em.sub.440/535] value statistically significant increased by 47.3 [+ or -] 5.4% (n = 19, p < 0.001). One [micro]M of [alpha]-hederin for 60 min. did not show any influence on the GRK2 activity. The corresponding [Em.sub.440/535] value was 1.05 [+ or -] 0.1 (n = 12) and comparable to the solvent control (Fig. 4A).

The direct influence of [alpha]-hederin on the PKA activity was determined with the Z'-Lyte[TM] kinase assay Kit Ser/Thr 1. The coumarin and fluorescein double-labeled protein (substrate) showed a statistically significant increase of the [Em.sub.440/535] value by a factor of 2.31 [+ or -] 0.12 (p < 0.001, n = 6) after maximum proteolytic cleavage, compared to the approach with 1.15 [micro]M PKA dissolved in 0.5% DMSO.

Compared to the pure PKA approach its activity was not influenced by DMSO (PKA control) (n = 5). Twenty [micro]M of the broad spectrum inhibitor staurosporine was used as positive control which led to a statistically significant increase of the [Em.sub.440/535] value by 76.7 [+ or -] 21.2% (n = 6, p < 0.001) compared to the activity of PKA dissolved in 0.5% DMSO. An inhibitory effect of 1 [micro]M [alpha]-hederin to PKA activity was not observed (n = 5) (Fig. 4B).


H. helix leaves dry extracts are used in the treatment of acute and chronic respiratory tract diseases associated with coughing (Gulyas et al, 1997; Hofmann et al., 2003; Kraft, 2004; Mansfeld et al., 1997, Mansfeld et al., 1998). Based on clinical studies and available scientific data they are classified by the European Committee on Herbal Medicinal Products (HMPC) for a "well established use" in the treatment of cough associated with viscous mucus and hypersecretion.

Although the mode of action of H. helix leaves dry extracts is not yet fully understood it has been shown to contain saponins e.g. [alpha]- and [beta]-hederin that enhance the [[beta].sub.2]AR responsiveness in A549 and HASM cells, respectively, which mediates the secretolytic and bronchospasmolytic effect (Greunke et al., 2015; Sieben et al., 2009). Recently Wolf et al. (2011) were able to show that pre-treatment with [alpha]-hederin improves isoprenaline-mediated relaxation of methacholine pre-contracted muscle strips of bovine trachea.

The responsiveness of many G protein-coupled receptors (GPCR) and thus also for the [[beta].sub.2]AR is regulated at conditions of high agonist concentrations by heterologous and homologous desensitization and subsequently by internalization.

In heterologous desensitization the [[beta].sub.2]AR is phosphorylated by the cAMP dependent PKA in the third intracellular loop or in the C-terminal region on Ser261/262 and Ser345/346 (Nobles et al., 2011). Phosphorylation leads to an uncoupling of the associated G protein and a switch of G protein association from [G.sub.s] to [G.sub.i] is also discussed (Benovic, 2002). The switch to [G.sub.i] would additionally antagonize the ligand induced receptor activation.

In homologous desensitization the [[beta].sub.2]AR is phosphorylated at several serines and threonines in the C-terminus by GRKs. GRKs are serine/threonine kinases with seven subtypes discovered so far (GRK1-7) (Penela et al., 2003). [[beta].sub.2]ARs in HEK293 cells are mainly associated to the subtypes 2 and 6 (GRK2/6) (Nobles et al., 2011). GRK2 is located in the cytosol and needs free [G.sub.[beta]y] dimers to associate with the membrane, thus in contrast to PKA it only phosphorylates activated receptors (Kohout and Lefkowitz, 2003). Phosphorylation by GRK2 induces uncoupling of G protein [G.sub.s[alpha]] and further promotes the binding of arresting proteins called arrestins (Kallal et al., 1998). So far four family members of [beta]-arrestins [[beta]-arr) have been found, of which [beta]-arr2 is predominant for [[beta].sub.2]ARs (Marchese et al., 2003). [beta]-arr2 binds to the GRK phosphorylated receptor and interacts with a dathrin adaptor protein (AP-2). The complex now enables clathrin to associate with the receptor and to build a clathrin coated pit in which the receptor is internalized via endocytosis (Claing et al., 2002; Marchese et al., 2003). Dependent to ongoing stimulation of the cell [[beta].sub.2]ARs can be relocated to the plasma membrane or degraded. It has been shown by Krasel et al. (2008) that the phosphorylation of several serines by GRKs in the cluster between Ser355 and Ser364 is essential for [beta]-arrestin binding and thus for [[beta].sub.2]AR internalization (Krasel et al., 2008).

Compared to [alpha]-hederin the structurally related [beta]-hederin is present only in very small amounts in the H. helix leaves dry extract, and therefore plays a minor role for the pharmacological effects (Greunke et al., 2015). For this reason, only [alpha]-hederin was investigated in the present study. The [alpha]-hederin mediated increase of [[beta].sub.2]-adrenergic responsiveness in A549 and HASM cells can be explained by inhibition of [[beta].sub.2]AR internalization and thereby an increased [[beta].sub.2]AR binding and formation of cAMP. Our findings on the influence of a- hederin on the PKA and GRK2 mediated phosphorylation of [[beta].sub.2]AR, respectively, lead to the conclusion that the inhibition of homologue desensitization is causal for the repression of [[beta].sub.2]AR internalization.

Since the ability to investigate phosphorylation level by In-Cell Western has been proven before by other research groups (Du et al., 2007), we performed own In-Cell Western experiments and were able to show a dose-dependent inhibition of the GRK2 mediated phosphorylation at Ser355/356 by [alpha]-hederin under stimulating conditions. As a positive control 0.5 [micro]M isoprenaline was used, which resulted in increased phosphorylation of GFP-[[beta].sub.2]AR of about 30.0 [+ or -] 4.1%. On the other hand Shi et al. (2011) found an increased phosphorylation of hemagglutinin (HA) tagged [[beta].sub.2]AR of about 60% in HEK293 cells for a comparable concentration of salbutamol. While for Shi et al. (2011) the extracellular N-terminus of [[beta].sub.2]AR was tagged with HA, in our experiments the intracellular C-terminus was tagged with GFP. Possibly the intracellular GFP-tag affects the interaction with GRK2, which could explain the observed lower isoprenaline mediated phosphorylation of GFP-[[beta].sub.2]AR. Moreover, salbutamol is a stronger agonist compared to isoprenaline, which also may lead to higher phosphorylation rates. One [micro]M [alpha]-hederin led to a decrease of 11.1 [+ or -] 5% in [[beta].sub.2]AR phosphorylation while lower concentrations (0.1 [micro]M and 0.5 [micro]M) did not show significant inhibition of GRK2 mediated phosphorylation. Remarkably, Shi et al. (2011) found a 30% inhibition of salbutamol mediated phosphorylation of HA-[[beta].sub.2]AR in HEK293 cells, however, using a significantly higher concentration of 100 [micro]M glycyrrhetic acid (GA), a structural related saponin from liqourice.

An influence of [alpha]-hederin on the PKA mediated phosphorylation of GFP-[[beta].sub.2]AR could not been determined.

We further performed FRET experiments in solution but did not find any direct effect of [alpha]-hederin on the phosphorylation potency of the two kinases. Thus it appears that the effect of [alpha]-hederin on GRK2 is mediated indirectly in cell experiments. Saponins are membrane-active substances that interact with membrane components such as phospholipids and cholesterol, and thereby modulate membrane dynamics and possibly functions of membrane associated proteins (Augustin et al., 2011; Lorent et al., 2014). Membrane integrated [alpha]-hederin could interfere in this way the interaction between [[beta].sub.2]AR and GRK2.

Our results agree with earlier findings of Shi et al. (2011) who showed that GA also enhances the [[beta].sub.2]AR responsiveness by GRK2 inhibition. They showed that GA reduces the plasma membrane cholesterol and thus changes the fluidity of plasma membranes leading to a release of raft-associated [G.sub.[alpha]s] which increases the [[beta].sub.2]AR-[G.sub.[alpha]s] coupling and subsequently decreases [[beta].sub.2]AR internalization (Shi et al., 2012). Although the used concentration of GA (100 [micro]M) is considerably higher than we applied for [alpha]-hederin (1 [micro]M), the findings on the [[beta].sub.2]AR phosphorylation seem to indicate a similar mode of action. Due to the lower dose used, one could assume that [alpha]-hederin seems to be even more potent in the GRK2 inhibition.

Further studies regarding the recruitment of [beta]-arr2 and GRK2 to [[beta].sub.2]AR could give information about how [alpha]-hederin inhibits the homologue desensitization.


In clinical studies secretolytic and bronchospasmolytic effects were described for Hedera helix leaves dry extracts. So far, these effects have been explained by a [alpha]-hederin mediated inhibition of [[beta].sub.2]AR internalization and subsequently an increase in [[beta].sub.2]-adrenergic responsiveness of the airways. Until now, the molecular mechanism responsible for the inhibition of [[beta].sub.2]AR internalization has not been described. The present paper indicates [alpha]-hederin as an indirect GRK2 inhibitor. By demonstrating a reduced homologous desensitization the [alpha]-hederin mediated internalization inhibition of [[beta].sub.2]AR under stimulating conditions can be explained. Thus, another important aspect in the mode of action of [alpha]-hederin as a pharmacological relevant ingredient of H. helix leaves dry extract could be clarified.


Article history:

Received 13 August 2015

Revised 17 November 2015

Accepted 4 December 2015

Conflict of interest

The authors have declared no conflict of interest. Acknowledgments

This work was supported by a research grant of Engelhard Arzneimittel GmbH & Co. KG, Niederdorfelden, Germany (grant no. PSP H-061.0037).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.12.001.


Augustin, J.M., Kuzina, V., Andersen, S.B., Bak, S., 2011. Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry 72, 435-457.

Benovic, J.L., 2002. Novel beta2-adrenergic receptor signaling pathways. J. Allergy Clin. Immunol. 110 (6 Suppl), S229-S235.

Claing, A., Laporte, S.A., Caron, M.G., Lefkowitz, R.J., 2002. Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and beta-arrestin proteins. Prog. Neurobiol. 66, 61-79.

Du. Y., Danjo, K., Robinson, PA, Crabtree, J.E., 2007. In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor. Microbes Infect. 9, 838-846.

Ferguson, S.S, 2001. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. 53, 1-24.

Giembycz, MA, Newton, R., 2006. Beyond the dogma: novel [[beta].sub.2]-adrenoceptor signaling in the airways. Eur. Respir. J. 27, 1286-1306.

Greunke, C., Hage-Htilsmann, A., Sorkalla, T., Keksel, N., Haberlein, F., Haberlein, H., 2015. A systematic study on the influence of the main ingredients of an ivy leaves dry extract on the [[beta].sub.2]-adrenergic responsiveness of human airway smooth muscle cells. Pulm. Pharmacol. Ther. 31, 92-98.

Gulyas, A., Repges, R., Dethlefsen, U., 1997. Konsequente Therapie chronisch obstruktiver Atemwegserkrankungen bei Kindern. Atemwegs- und Lungenkrankheiten 23, 291-294.

Hofmann, D., Hecker, M., Volp, A., 2003. Efficacy of dry extract of ivy leaves in children with bronchial asthma--a review of randomized controlled trials. Phytomedicine 10, 213-220.

lino, M., Furugori, T., Mori, T., Moriyama, S., Fukuzawa, A., Shibano, T., 2002. Rational design and evaluation of new lead compound structures for selective betaARKl inhibitors. J. Med. Chem. 45 2150-2149.

Johnson, M., 1998. The beta-adrenoceptor. Am. J. Respir. Crit. Care Med. 158 (5 Pt 3), S146-S153.

Kallal, L., Gagnon, A.W., Penn, R.B., Benovic, J.L., 1998. Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged beta2-adrenergic receptor. J. Biol. Chem. 273, 322-328.

Kohout, T.A., Lefkowitz, R.J., 2003. Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol. Pharmacol. 63, 9-18.

Kraft, K., 2004. Vertraglichkeit von Efeublattertrockenextrakt im Kindesalter. Z. Phythother. 25, 179-181.

Krasel, C., Zabel, U., Lorenz, K., Reiner, S., Al-Sabah, S., Lohse, M.J., 2008. Dual role of the beta2-adrenergic receptor C terminus for the binding of beta-arrestin and receptor internalization. J. Biol. Chem. 283, 31840-31848.

lochner, A., Moolman, J.A., 2006. The many faces of H89: a review. Cardiovasc. Drug Rev. 24, 261-274.

Lorent, J.H., Quetin-Leclercq, J., Mingeot-Leclercq, M.P., 2014. The amphiphilic nature of saponins and their effects on artificial and biological membranes and potential consequences for red blood and cancer cells. Org. Biomol. Chem. 12, 8803-8822.

Mansfeld, H.J., H8hre, H., Repges, R., Dethlefsen, U, 1997. Sekretolyse und Bronchospasmolyse--Klinische Studie: Behandlung von Kindern mit chronisch obstruktiven Atemwegserkrankungen. TW PSdiatrie 10, 155-157.

Mansfeld, H.J., Hohre, H., Repges, R., Dethlefsen, U., 1998. Therapie des Asthma bronchiale mit Efeublatter-Trockenextrakt. MMW Fortsch. Med. 140, 26-30.

Marchese, A., Chen, C., Kim, Y.M., Benovic, J.L., 2003. The ins and outs of G protein-coupled receptor trafficking. Trends Biochem. Sci. 28, 369-376.

Nobles, K.N., Xiao, K., Ahn, S., Shukla, A.K., Lam, C.M., Rajagopal, S., ..., Lefkowitz, R.J., 2011. Distinct phosphorylation sites on the [[beta].sub.2]-adrenergic receptor establish a barcode that encodes differential functions of [beta]-arrestin. Sci. Signal 4 (185), ra51. doi:10.1126/scisignal.2001707.

Penela, P., Ribas, C., Mayor Jr., F., 2003. Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases. Cell Signal 15, 973-981.

Ruegg, U.T., Burgess, G.M., 1989. Staurosporine, K-252 and UCN-01: potent but nonspecific inhibitors of protein kinases. Trends Pharmacol. Sci. 10, 218-220.

Shi, Q,, Hou, Y., Yang, Y., Bai, G., 2011. Protective effects of glycyrrhizin against beta(2)-adrenergic receptor agonist-induced receptor internalization and cell apoptosis. Biol. Pharm. Bull. 34, 609-617.

Shi, Q,, Hou, Y., Hou, J., Pan, P., Liu, Z., Jiang, M., Gao, J., Bai, G., 2012. Glycyrrhetic acid synergistically enhances ^-adrenergic receptor-Gs signaling by changing the location of Galphas in lipid rafts. PLoS One 7, e44921.

Sieben, A., Prenner, L, Sorkalla, T., Wolf, A., Jakobs, D., Runkel, F., Haberlein, H., 2009. [alpha]-Hederin, but not hederacoside C and hederagenin from Hedera helix, affects the binding behaviour, dynamics, and regulation of beta 2-adrenergic receptors. Biochemistry 48, 3477-3482.

Tran, T.M., Friedman, J., Qunaibi, E., Baameur, F., Moore, R.H., Clark, R.B., 2004. Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the beta2-adrenergic receptor using phosphoserine-specific antibodies. Mol. Pharmacol. 65, 196-206.

Wolf, A., Gosens, R., Meurs, H., Haberlein, H., 2011. Pre-treatment with [alpha]-hederin increases [beta]-adrenoceptor mediated relaxation of airway smooth muscle. Phytomedicine 18, 214-218.

Janka Schulte-Michels, Anne Wolf Stefan Aatz, Katharina Engelhard, Anne Sieben, Manuel Martinez-Osuna, Felix Haberlein, Hanns Haberlein *

Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelms-University of Bom, Bom, Germany

Abbreviations: [[beta].sub.2]AR, [[beta].sub.2]-adrenergic receptor; PKA, protein kinase A; GRK2, G protein-coupled receptor kinase 2; GFP, green fluorescent protein; FRET, Forster resonance energy transfer.

* Corresponding author. Tel.: 228 736555; fax: 22 8 732416.

E-mail address: (H. Haberlein).
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Author:Schulte-Michels, Janka; Aatz, Anne Wolf Stefan; Engelhard, Katharina; Sieben, Anne; Martinez-Osuna,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Jan 15, 2016
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